cancers Review PARP Inhibitors as Therapeutics: Beyond Modulation of PARylation Ahrum Min 1,2 and Seock-Ah Im 1,2,3,4,* 1 Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea; [email protected] 2 Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea 3 Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Korea 4 Translational Medicine, Seoul National University College of Medicine, Seoul 03080, Korea * Correspondence: [email protected]; Tel.: +82-2-2072-0850; Fax: +82-2-765-7081 Received: 30 December 2019; Accepted: 5 February 2020; Published: 8 February 2020 Abstract: Poly (ADP-ribose) polymerase (PARP) 1 is an essential molecule in DNA damage response by sensing DNA damage and docking DNA repair proteins on the damaged DNA site through a type of posttranslational modification, poly (ADP-Ribosyl)ation (PARylation). PARP inhibitors, which inhibit PARylation through competitively binding to NAD+ binding site of PARP1 and PARP2, have improved clinical benefits for BRCA mutated tumors, leading to their accelerated clinical application. However, the antitumor activities of PARP inhibitors in clinical development are different, due to PARP trapping activity beyond blocking PARylation reactions. In this review, we comprehensively address the current state of knowledge regarding the mechanisms of action of PARP inhibitors. We will also discuss the different effects of PARP inhibitors in combination with cytotoxic chemotherapeutic agents regarding the mechanism of regulating PARylation. Keywords: PARP; PARP inhibitors; PARylation; trapping; cancer therapeutic strategy 1. Introduction All cells have more than tens of thousands of events that damage DNA in multiple ways, ranging from single base mismatches, bulky adducts in DNA bases, intra- and inter-strand DNA crosslinks, to single- and double-strand breaks (SSBs and DSBs) [1,2]. This DNA damage threatens genomic stability. There are DNA damage responses (DDRs), the sophisticated mechanisms of genome protection in cells, that function to activate the cell cycle checkpoint pathway to maintain genome stability by stopping or delaying the cell cycle during DNA damage or unstable DNA replication to allow the repair of damaged DNA lesions. DDRs activate transcription of a repair molecule or pro-apoptotic molecule to cause overexpression of the related molecule. DDRs activate mechanisms to remove uncontrolled damaged cells and to repair DNA damage from apoptosis caused by chromatid instability [3]. Regulation of the DDR pathway is induced by post-translational modification (PTM); poly ADP-Ribosylation (PARylation) is the pivotal PTM that occurs rapidly at the damage site during DDR [4,5]. PARylation is the reaction of transferring ADP-ribose residues to target substrates by ADP-ribosyl transferase using NAD+. It rapidly recognizes multiple types of DNA damage, including SSBs, and is recruited to the damaged site to induce the recruitment of DDR molecules so that the poly (ADP-Ribose) polymerase (PARP), specifically PARP1, PARP2, PARP3, PARP5a, and PARP5b, which are known as the major molecules of DDR, performs poly (ADP-ribose) (PAR) synthesis in humans [6]. PARP1 was proposed as a new treatment for cancer, as the synthetic lethality concept suggested that its depletion in breast-cancer patients with germline mutations in the BRCA1 or BRCA2 genes, key molecules in the homologous recombination (HR) pathway, could cause cancer cell death [7,8]. Since it was proven to Cancers 2020, 12, 394; doi:10.3390/cancers12020394 www.mdpi.com/journal/cancers Cancers 2020, 12, 394 2 of 16 be true, PARP inhibitors that inhibit DDR resulted in improved clinical benefits and became standard therapy [9–11]. To date, four PARP inhibitors have been approved by the FDA and are being applied clinically. However, while all PARP inhibitors inhibit PARP catalytic activities, they have different cytotoxicities. Therefore, the anti-tumor effects of the PARP inhibitors have been suggested to be due to PARP trapping, as well as the inhibition of the enzymatic activities [12,13]. The catalytic inhibition and trapping effects of PARP are tightly regulated, and the cytotoxicity of each mechanism can cause different reactivities. Therefore, in this review, based on mechanisms of PARP, we intend to examine the difference of anti-tumor effect of the PARP inhibitors and the current aspect of the roles in combination treatment. 2. PARPs and PARylation Poly (ADP-ribose) polymerase (PARP) is a family of 17 proteins in mammals, encoded by different genes, but with a conserved catalytic domain. Other than the catalytic domain, PARP family members contain one or more other motifs or domains, including zinc fingers, a breast cancer-susceptibility protein (BRCA) C-terminus-like (BRCT) motifs, ankyrin repeats, macro domains, and WWE domains [14] (Figure1A). PARP1 was the first family member identified and has a critical role in SSB repair through the metabolism of recruiting and dissociating repair proteins by PARylation. In addition to DNA damage repair, PARP1 has important roles in a various range of cellular processes from cell proliferation to cell death, due to having diverse substrates like nuclear proteins involved in transcriptional regulation, apoptotic cell death, chromatin decondensation, inflammation, and cell cycle regulation [15,16]. PARP1 has a total molecular weight of 113 kDa and contains seven independent domains (Figure1B) [ 5,17]. The N-terminus is the DNA binding domain (residues 1-353), which contains three zinc-finger DNA-binding domains, ZnFI, ZnFII, and ZnFIII, which are responsible for recognizing sites of damaged DNA and binding through allosteric activation. In the N-terminus there is a nuclear localization sequence (NLS) that places PARP1 in the nucleus with the KRK-X(11)-KKKSKK sequence. Between residues 211 and 214, there is a DEVD site that is cleaved by caspase into fragments of 23 and 89 KDa during apoptosis [18]. Residues 373 to 662 are the auto-modification domain consists of BRCA C-terminus-like (BRCT) domain serving sites of auto-ADP ribosylation and functioning in protein-protein interaction, and a WGR domain which roles in activating DNA damage repair by interaction with ZnFI, ZnFII, and catalytic domain. The auto-modification domain is rich in glutamate and lysine residues and is the site of self-PARylation. Finally, the C-terminus (residues 662–1014) is the catalytic domain, and the (ADP-Ribosyl) transferase (ART) domain is a NAD+ acceptor site where the His-Try-Glu residues called ART signatures are preserved well [19–21]. The helical subdomain (HD), an auto-inhibitory domain in the C-terminus, inhibits the binding of PARP1 and β-nicotinamide adenine dinucleotide without binding to DNA. When PARP1 binds to the DNA damage site, the auto-inhibitory function of HD is removed. The activation of the catalytic activity of ART and the generation of PAR chains in the target protein lead to the recruitment of DNA repair molecules. Thereafter, PARP1 is dissociated from DNA by auto-PARylation of PARP1, resulting in DNA repair [22]. Cancers 2020, 12, 394 3 of 16 Cancers 2020, 12, x FOR PEER REVIEW 3 of 17 Figure 1. PARPs structure (A) The PARP family consists of 17 members, divided into five subgroups Figure 1. PARPs structure (A) The PARP family consists of 17 members, divided into five subgroups according to domain structure and function: DNA damage-dependent PARPs (PARP1, PARP2, and according toPARP3), domain tankyrases structure (tankyrase1/PARP5 and function: and DNAtankyrase2/PARP5b), damage-dependent CCCH-type PARPs PARPs (PARP1, (PARP7, PARP2, and PARP3), tankyrases (tankyrase1/PARP5 and tankyrase2/PARP5b), CCCH-type PARPs (PARP7, PARP12, and PARP13), macro-PARPs [B-aggressive lymphoma 1 (BAL1)/PARP9, BAL2/PARP14, and BAL3/PARP15], and other PARPs (PARP4, PARP6, PARP8, PARP10, PARP11, and PARP16). The catalytic domain at the C-terminus is conserved in all members and contains additional zinc fingers, BRCA C-terminus-like (BRCT) motifs, ankyrin repeats, macro domains, and WWE domains. (B) The seven major domains of PARP1 include three zinc-finger domains in the DNA binding domain, the BRCT domain in the auto-modification domain, and the pADPr accepting WGR domain (W), located centrally. The C-terminus has two catalytic domains: ART and a helical domain (HD). Cancers 2020, 12, 394 4 of 16 This series of reactions is caused by PARylation. While the catalytic domain is conserved in the PARP family, only PARP1/2/3/4/5a/5b activates PARylation by possessing the His-Tyr-Glu motif called the “ART signature” [5,15,23–26]. The role of PARP3 as an (ADP-ribosyl) transferase is controversial. PARP4 is the largest protein in the PARP family, and PARP5a and PARP5b, classified as tankyrase1/2, have a SAM (Sterile Alpha motif) domain that interacts between proteins with the ability to homo- and hetero-oligomerize PARP1 and 2. PARP1 transfers ADP-ribose residues from NAD+ to acidic amino acid residues such as glutamates (E), lysine (K), arginine (R), serine (S), and aspartate (D), forming the negative poly (ADP-ribose) (PAR) chain [5,24,26]. PARP1 is believed to perform more than 90% of total PARylation in response to DNA damage. As soon as DNA damage occurs, ADP-ribosylation is covalently bound to the carbonyl group of the acidic residues of the target protein via ester bonds. PARP then forms a PAR chain by cleaving the glycosidic bond between nicotinamide and ribose of NAD+ by catalytic activity and binding ADP-ribosylation to the